Notes

MARINE MAMMAL SCIENCE, 32(2): 753–764 (April 2016) Published 2015. This article is a U.S. Government work and is in the public domain in the USA DOI: 10.1111/mms.12275

Characterization of the putatively introduced red alga Acrochaetium secundatum (Acrochaetiales, Rhodophyta) growing epizoically on the pelage of southern sea otters (Enhydra lutris nereis)

GENA B. BENTALL,1,2 U.S. Geological Survey, Western Ecological Research Center, Santa Cruz Field Station, 100 Shaffer Road, Santa Cruz, California 95060, U.S.A.; BARRY H. ROSEN, U.S. Geological Survey, 12703 Research Parkway, Orlando, Florida 32826, U.S.A.; JESSICA M. KUNZ and MELISSA A. MILLER, California Department of Fish and Wildlife, Marine Wildlife Veterinary Care and Research Center, 1451 Shaffer Road, Santa Cruz, Califor- nia 95060, U.S.A.; GARY W. SAUNDERS, University of New Brunswick PO Box 4400, Fred- ericton, New Brunswick E3B 5A3, Canada; NICOLE L. LAROCHE,3 U.S. Geological Survey, Western Ecological Research Center, Santa Cruz Field Station, 100 Shaffer Road, Santa Cruz, California 95060, U.S.A.

Ecological associations between epibionts (organisms that live on the surface of another living organism) and vertebrates have been documented in both marine and terrestrial environments, and may be opportunistic, commensal, or symbiotic (Lewin et al. 1981, Holmes 1985, Allen et al. 1993, Bledsoe et al. 2006, Pfaller et al. 2008, Suutari et al. 2010). Although epibiont proliferation is frequently reported on slow-moving, sparsely haired organisms such as manatees and sloths, reports from densely furred, highly mobile mammals are much less common. There are reports of epizoic algae for several species of pinnipeds (Kenyon and Rice 1959, Scheffer 1962, Baldridge 1977, Allen et al. 1993), which rely to varying degrees on both pelage and blubber for thermoregulation, but the phenomenon has not been widely described. Scheffer (1962) noted that red algae was fairly common on the pelage of northern fur seals (Callorhinus ursinus), pinnipeds for which fur likely makes a comparatively high contribution to thermoregulation (Donohue et al. 2000). For species with pelage that plays a critical role in thermal insulation, it seems implausible that an epibiont would persist on healthy individuals that devote sig- nificant energy resources toward grooming and actively maintaining their coat. Biological

1Corresponding author (e-mail: [email protected]). 2Current address: 6901 Chenango Court, Goleta, California 93117, U.S.A. 3Current address: 1961 Main Street, #199, Watsonville, California 95076, U.S.A.

753 754 MARINE MAMMAL SCIENCE, VOL. 32, NO. 2, 2016 characteristics of epibiont settlement and attachment, and physiological requirements of epizoic species play key roles in their successful colonization and potential host impacts. To investigate this relationship, we explore a novel discovery of an epizoic alga from southern sea otters, including describing algal development on sea otter hair and molecu- lar identification of the algae. Sea otters are highly active marine mustelids that rely on their fur for insulation in the cold waters of the northeast Pacific Ocean. Sea otters have the densest fur coat of any mammal, with interlocking hairs and sebaceous gland secretions that create an insulating layer of air that prevents heat loss by minimizing contact between the skin surface and cold seawater (Williams et al. 1992). If the pelage becomes saturated with water, otters can quickly succumb to hypothermia (Costa and Kooyman 1982). In order to maintain this critical thermal barrier, sea otters devote between 5% and 15% of their daily activity budget to vigorous grooming of their fur (Riedman and Estes 1990, Yeates et al. 2007). The sea otter’s frequent grooming and potential neg- ative impacts of hair contamination on insulating properties of the pelage and sur- vival seems incompatible with successful colonization of hairs by epizoic species. However, recent observational studies of live, tagged otters and postmortem exami- nations have documented growth of firmly attached, filamentous red algae on the pelage of numerous southern sea otters (Enhydra lutris nereis). To begin to assess basic trends and prevalence for epizoic algae on this species, we reviewed records from live sea otter tagging operations conducted by the U.S. Geo- logical Survey, Monterey Bay Aquarium, and California Department of Fish and Wildlife. These operations took place from 2000 through 2014, and spanned the entire southern sea otter range from Santa Cruz to Santa Barbara counties. During these studies, approximately 550 sea otters were captured and immobilized for tag- ging, VHF transmitter implantation, sample collection, morphometric measurement, and health examination (Tinker et al. 2008). Detailed data on physical condition were recorded for each individual, with observations of attached algae noted at the discretion of the recorder, rather than as a standardized component of the record. As a result, these retrospective capture records likely underestimate spatial and temporal trends for epizoic red algal growth on sea otters. Tagged otters were monitored using telemetry techniques and high-powered tele- scopes, 1–7 times weekly for <1to>10 yr, depending on the battery life of the trans- mitter, retention of flipper tags, and animal survival. Initial observations of attached algae were most often made while the otter was examined under sedation during the tagging process, although some observations were postrelease during field monitoring efforts. Individual animal survival was assessed by calculating the time from first observation of algae until the last resight record. Photo-documentation of tagged otters with visible attached algae was performed opportunistically when animals were within camera range, typically 20–50 m from the observer. In addition to examining/monitoring attached algae on live otters, we conducted a retrospective review of southern sea otter stranding records from 1997 through 2014 for recorded observations of algal growth. In addition to the review of all stranding records of dead southern sea otters during that time period, we compiled observations of attached algae from sea otter necropsies performed at the California Department of Fish and Wildlife, Marine Wildlife Veterinary Care and Research Center (MWVCRC) in Santa Cruz, California, from 2003 through February 2015. The depth and detail of postmortem examination was dependent upon each animal’s post- mortem state (fresh dead and tagged study animals received more thorough examina- tions), but all recovered southern sea otter carcasses were necropsied during this time NOTES 755 period. Necropsy examinations facilitate more thorough assessment of the distribu- tion and extent of attached algae, assessment of nutritional condition, and documen- tation of any concurrent health conditions, when compared with field observations. Algae adhering to the fur of sea otter carcasses was not systematically assessed or recorded during necropsy prior to late 2014. Thus, retrospective necropsy data likely underestimates spatial and temporal trends for epizoic red algal growth on sea otters. For the purpose of this review, we included only fresh and moderately decomposed cases with reports of attached algae, because algal growth was not likely to be detected on the fur of markedly decomposed animals. Because algal chlorophyll and other endogenous pigments often fluoresce when exposed to ultraviolet light, ultraviolet illumination can be used to enhance detection of algal blooms (Boney 1972). To assess the utility of this technique to detect algal growth on sea otter fur, we scanned the pelage of a subset of necropsy cases in a darkened room using a hand-held, long-wave UV lamp (Blak-Ray Model UVL 56, 365 nm). To distinguish algal growth on sea otter fur from petroleum contamination, we also assessed fluorescence patterns of selected refined and nonrefined petroleum com- pounds (e.g., commercial grease and motor oil, and unrefined, weathered tar originat- ing from natural underwater petroleum seeps along the central California coast). The commercial products were applied manually to the pelage just prior to necropsy, while the weathered tar patties were naturally occurring petroleum compounds that were observed on the fur of a few stranded southern sea otters. The color and relative brightness of fluorescence of petroleum compounds was subjectively compared with fluorescence from microscopically confirmed algal growth. Samples of fur with attached algae were also collected opportunistically at sea otter necropsies for microscopic examination of morphological features, and to facilitate taxonomic identification of the epibiont. Individual hairs were mounted in water, cover-slipped, and examined on an Olympus BX-51 microscope using differential interference microscopy. Finally, we prepared a subset of the material for genetic analysis by drying in silica gel. Algal samples from five different otter carcasses were submitted for DNA bar code analyses (Table S1). Extraction of DNA and amplification of COI-5P followed Saunders and McDevit (2012), while rbcL amplification followed Saunders and Moore (2013). Amplified products were sequenced at Genome Quebec (http://www.genome quebec.com) with consensus sequences generated in Geneious 8.1.3 (Biomatters Ltd., San Francisco, CA) and searched through BOLD and GenBank to confirm the iden- tity of the red alga. Based on retrospective review, the earliest anecdotal observations of red algal growth on fur of wild southern sea otters date back to the 1980s,4 while the first writ- ten record from a live otter was from an adult male captured in central California in October 2002. External algal growth was described for eight sea otters during capture and tagging efforts (three males, five females) spanning the entire southern sea otter range between 2002 and 2014. All eight otters were prime-aged adults (estimated age from 3 to 10 yr old) that appeared to be in good health. Following capture and tagging, seven of the eight otters with visible epizoic red algal growth survived at least one year (0.87). This survival rate is consistent with the population-wide sur-

4Personal communication from J. Ames, California Department of Fish and Wildlife (retired), 1451 Shaffer Road, Santa Cruz, California 95060, U.S.A., 1 March 2015. 756 MARINE MAMMAL SCIENCE, VOL. 32, NO. 2, 2016 vival probability for prime-aged adult southern sea otters (0.75–0.78 for males, 0.86–0.88 for females) (Tinker et al. 2006), suggesting that epizoic algal growth did not influence survival. Algal patches were observed on three additional tagged otters (one male, two females) during postcapture monitoring, and all three survived at least 1 yr after documentation of algal growth. While algae may attach to hair on any part of the otter’s body, it was most frequently noted on the tail, dorsal head, shoulders, and perineum and was most often observed on otters with lightened (grizzled) fur extending beyond the crest of the head (Fig. 1). In one case, a tagged territorial male (998-05) was observed repeatedly with attached algae over 2 yr, and the density of an easily visible algal patch on the dorsal head fluctuated over time. The presence of attached algae on 998-05 was first noted during routine telescope-based observations during August and September of 2010, and coincided with a period of reduced body condition (visibly emaciated) and deviation of activity, characterized by decreased for- aging, grooming, swimming, and social interactions from average levels. During this period, this male was also utilizing habitat closer to shore than usual, and we cannot rule out the possibility that close proximity facilitated detection of algae. Observa- tions made 2 yr later (September 2012), after this animal’s nutritional condition and activity levels had normalized, document the persistence and, in fact, increased den- sity of the algae patch. Interestingly, when 998-05 was necropsied 8 mo later (May 2013), no algae was noted, suggesting that the algal density had decreased to a level that was difficult to detect. It is important to note that proximity to shore and exten- sive grizzling of the fur made detection of algae more probable in the case of 998-05, whereas in most cases distance and submersion make detection difficult in free-rang- ing otters. Epizoic red algal growth was first noted on fur of necropsied southern sea otters in 2003, with additional reports spanning from 2003 through April 2015. Of 1,240 fresh or moderately decomposed sea otter necropsy records examined from 2003 through February 2015, 52 noted adherent algae, and 50 highlighted the distribu- tion of algal growth on the otter’s body, including written descriptions, diagrams, and photographs. The distribution of algal growth for necropsy cases is similar to observational data for live otters, most frequently including the tail and perineum (n = 44, 88% of 50 cases with descriptions), hind limbs (42%), front limbs (22%),

Figure 1. Wild sea otter with visible epizoic red algae, indicated by yellow bracket. Photo credit: B. Hatfield. NOTES 757 lateral body wall near the water line (20%), ventral chest and abdomen (16%), dor- sum (10%), and head or neck (10%). One reliable indicator of nutritional condition in sea otters is the amount and distribution of subcutaneous adipose (Kreuder et al. 2005). During necropsy, subcutaneous adipose stores were ranked from 0 (none), to 1 (scant), 2 (fair), 3 (good), and 4 (abundant). Sea otter necropsy cases with adherent algae spanned the nutritional range (mean = 1.38, SD = 1.53), suggesting that epi- zoic red algal growth was not correlated with progressive nutritional decline prior to death. Ultraviolet illumination of putative red algal growth on sea otter fur at necropsy elicited brilliant orange fluorescence (Fig. 2A, B), facilitating algal detection, even on darker portions of the pelage. Ultraviolet illumination also revealed patches of algal growth that were too small, and/or too dispersed to be easily discernible under ambient light, thus enhancing detection. In contrast, refined petroleum products such as grease and motor oil reflected pale, gray-blue fluorescence on sea otter fur under ultraviolet illumination (Fig. 2 C), facilitating discernment between surface algal growth and petroleum contamination. Finally, we did not observe any fluores- cence for sea otter fur contaminated by nonrefined, weathered tar patties, such as those that emanate from underwater petroleum seeps along the central California coast. Microscopic examination of the epizoic alga (Fig. 3) revealed colonization and growth typical of the red algal genus Acrochaetium (Clayden and Saunders 2014). The initial cells to encounter and attach to fur appear to be monospores, nonmotile asex- ual spores that rely on water currents and possibly (in the case of mobile sea otters) direct contact for transport. Once attached, monospores undergo a series of divisions to produce monostromatic basal discs (Fig. 3A). Central cells of the basal discs pro- duce erect filament initials (Fig. 3B, arrows), which undergo transverse divisions (Fig. 3C, arrow) to yield short, unbranched, erect filaments (Fig. 3D). Ultimately, erect filaments (cells with apparent remnant of a central pyrenoid; Fig. 3E, arrow- heads) were slightly curved, secundly branched, and produced sessile or stalked monosporangia (Fig. 3E, arrows). Individual hair tips can be completely surrounded by branching masses of these filaments (Fig. 3F), which are visible without magnifi- cation. Genetic analysis with two widely applied DNA bar code markers (mitochondrial COI-5P [n = 2] and plastid rbcL[n = 5]; see Saunders and McDevit 2012, Saunders and Moore 2013) consistently assigned the specimens to the red algal species Acro- chaetium secundatum, Acrochaetiales (Table S1). The morphological characteristics described above (Fig. 3) are also consistent with this identification (Clayden and Saunders 2014). This species is common and widely distributed on a variety of algal and invertebrate hosts in the North Atlantic Ocean (Clayden and Saunders 2014). However, the first verified record for this species from the Northeast Pacific Ocean was from July, 2003 (Clayden and Saunders 2014). This species may have been intro- duced to the Northeast Pacific, as it was not reported in earlier detailed monographs by Drew (1928) and Garbary et al. (1982) for California and British Columbia, respectively. Many species of filamentous red algae are common in habitats that are physically challenging, such as rock or wood substrata of ocean or freshwater splash zones (Lin and Blum 1977). Red algae use a variety of strategies to manage desiccation and can withstand intermittent air exposure (Kain and Norton 1990). They are common epi- phytes of other algae, vascular plants, invertebrate shells, and other animals (Kain and Norton 1990). Extracellular mucilage secreted by algal spores enables them to 758 MARINE MAMMAL SCIENCE, VOL. 32, NO. 2, 2016

Figure 2. Enhanced detection of epizoic algae (A, B) during necropsy using normal interior lighting (A) and under UV-A light (B) revealing fluorescence. (C) The blue-gray fluorescence of petroleum-products (top: petroleum-based motor oil, bottom: petroleum-based grease) is shown for comparison. Photo credit A, B: G. Bentall, C: M. Miller. NOTES 759

Figure 3. The red alga, Acrochaetium secundatum, colonizing the fur of a southern sea otter (Enhydra lutris nereis). (A) Monospores undergo several divisions, forming monostromatic basal discs (B) that produce erect filament initials (arrow). The erect filament initials undergo trans- verse divisions, (C, arrow) eventually producing short unbranched filaments. Unbranched filaments were densely packed onto a hair (D), ultimately becoming slightly curved and secundly branched, producing sessile and stalked monosporangia (E, arrow). Note apparent remnant of a central pyrenoid (E, arrowheads). The mass of algae completely surrounds the tip of a hair (F). adhere to these various substrata (Pueschel 1990). Once the spores of A. secundatum come in contact with a surface, such as otter fur, they can adhere tightly to the sub- strata and survive shear stresses associated with the force of grooming. After the alga attaches, the diminutive nature of this species, with its short filaments (usually <5 mm), make it difficult to dislodge from the hair surface. Sea otters molt gradually throughout the year (Riedman and Estes 1990) so while the algae may remain firmly attached, the hairs with attached algae may be lost, reducing algal cover in the absence of local spread or new recruitment. An understanding of the morphology of the sea otter pelage and relative contribu- tion of each fur strata to insulation may provide insight into potential impacts of 760 MARINE MAMMAL SCIENCE, VOL. 32, NO. 2, 2016 attached algae on thermoregulation. The sea otter pelage is composed of two hair types that differ significantly in size and structure (Williams et al. 1992). Guard hairs are longer, of larger diameter, and more sparsely distributed, and likely function by supporting the underfur and channeling sea water away from this critical insulating layer. Finer, shorter, and denser hairs of the underfur interlock due to their scale pat- tern and waviness, forming an insulating air barrier between sea water and skin. In the current study, we observed red algal colonization on both underfur and guard hairs (although growth appeared to be more common on guard hairs). How- ever, algal growth and proliferation appeared to be restricted to the portion of each hair protruding above the insulation layer (Fig. 4). Presumably, algal growth requires continuous access to light, moisture, and nutrients, thus restricting colo- nization to the outer portion of hairs external to the air space of the insulation layer. Thus, insulating properties of the underfur appear to remain essentially undisturbed, and any impacts on hair function are confined to the pelage surface. Our study found no compelling evidence that attached algae compromise the sea otter’s ability to thermoregulate. Successful colonization of any host by algal epibionts depends on the suitability of host substrate relative to the colonizing organism’s requirements for space, shelter, light, and nutrients. As a marine mammal that spends much of its time floating at the surface in near shore, coastal marine habitat, the sea otter may provide a substrate remarkably similar to others colonized by the alga in intertidal habitats. For A. secun- datum, the dense pelage of sea otters provides a substratum with a high degree of habitat complexity. In addition to providing ample surface area for colonization and attachment, sea otter fur substrate may provide physical conditions well-suited for this species, plus protection from herbivory. Adapted to periodic exposure to wave activity and air, A. secundatum may survive by being resistant to removal from its sub- strate and is desiccation-tolerant, allowing it to remain attached to hair despite vigor- ous grooming, and undamaged by drying while the otter rests at the surface.

Figure 4. Longitudinal view of sea otter underfur and guard hairs, demonstrating concen- tration of algal growth on the outer portions of sea otter guard hairs with the greatest exposure to seawater and nutrients. Photo credit: M. Miller. NOTES 761

The distribution of A. secundatum on sea otter fur appears to suggest that the water-air margin at the point where the otter would naturally float at the surface is especially favorable for colonization, and may represent a region of optimal exposure to moisture, light, and air. The mobility of sea otters may also confer a selective advantage, in that algae are less likely to encounter invertebrate grazers that are com- mon on less mobile substrata. Sea otters may also provide direct nutritional benefits. In most coastal waters, growth of autotrophic algae is limited by access to nitrogen (Howarth and Marino 2006). When attached to sea otters, A. secundatum may adapt to utilizing organic nitrogen, such as host urea and amino acids, providing a continuous source for algal growth and reproduction. Our necropsy data revealed the anatomic areas most com- monly colonized by epizoic red algae were the perineum and ventral tail; areas in close proximity to excreted sea otter waste products. Colonization of the perineal region may also be correlated with modes of transference. The limited motility of monospores normally relegates their dispersal to the movement of water; however, on a host such as sea otters transference may also occur by direct contact during social, reproductive or maternal interactions, and frequent presence of algae around the per- ineum could suggest transmission during copulation. The relationship between epizoic algae and sea otters is dependent on both the extent to which the host provides suitable habitat for the colonizer, and the degree to which the colonization affects the health and survival of the host. While A. secunda- tum appears to be well equipped to survive efforts at removal by sea otter grooming, we do not know if the frequency and vigor of an individual otter’s grooming behavior affects the amount (abundance and density) of algal growth, nor do we know if speci- fic environmental conditions are more favorable for A. secundatum attachment, sur- vival, and proliferation. Our observations of changes in the extent of attached algae over time on tagged male 998-05 may exemplify response of the algae to changing environmental conditions or host behavior. If attached algae undergo uncontrolled proliferation, it is possible that pelage fouling could occur beyond a level that is con- sistent with adequate thermoregulation and survival. We also have yet to understand the extent to which extensive algal growth might affect hydrodynamics; it is possible that increased drag caused by an extensive algal load could reduce swimming and div- ing efficiency, causing the sea otter to incur additional energetic costs. Similarly, the presence of algae on the pelage may require the sea otter to increase time, effort and energy allocated to grooming. Given the paucity of records of A. secundatum in the Pacific Ocean prior to 2003, its growth on sea otters along the California coast may be an accidental product of algal introduction through ballast water or other anthropogenic mechanisms. Although sea otter research projects similar to those in California have been con- ducted in Alaska, Washington, and Canada, we only encountered three records of epizoic algae from a single tagging event in British Columbia in 2010,5 with all other records restricted to California. Although the timing of epizoic algal coloniza- tion of sea otter populations is uncertain due to inconsistent recording protocols, observations of algal growth on otters appear to have become more common over the past decade, although we cannot rule out the possibility that observations have increased due to enhanced awareness, rather than increased prevalence. In-depth

5Personal communication from K. A. Kloecker, 4210 University Drive, Anchorage, Alaska 99508- 4626, U.S.A., 12 June 2014. 762 MARINE MAMMAL SCIENCE, VOL. 32, NO. 2, 2016

examination of historic accounts and archival pelts may further clarify relationships between wild sea otters and the putatively introduced alga. If this is a relatively novel interaction, the sea otter and alga may be in the early stages of a coevolutionary relationship. It is likely that A. secundatum and other unrecognized epibiont algae have colonized the fur of more sea otters than our data indicate; enhanced detection of cryptic cases using UV illumination may reveal a higher frequency than was previously recog- nized, and this method should be included as part of sea otter physical examinations and necropsies. Questions regarding spatial, temporal, and seasonal patterns of occur- rence, and associations between specific sea otter behavioral types (i.e.,shallowvs. deep water foraging) or dietary preferences, and algal colonization remain unan- swered. Establishing interagency protocols for recording occurrence of epizoic algae on sea otters will facilitate future explorations of these trends.

Acknowledgments

We thank Dr. Martin Tim Tinker of the U.S. Geological Survey Western Ecological Research Center at the Santa Cruz field station for contributing data and advice. Thanks to Michelle Staedler and Jessica Fujii of the Monterey Bay Aquarium Sea Otter Research and Conservation department for contributing data and their extensive knowledge about wild sea otters. The University of Santa Cruz Institutional Animal Care and Use Committee approved the tagging research under permit #Tinkt1309, and tagging and subsequent monitoring was conducted under U.S. Fish and Wildlife Service permit #MA672624 (USGS) and #MA032027 (Monterey Bay Aquarium). Thanks to Ben Shaw, Erin Dodd, Francesca Batac, Michael Harris, and Colleen Young from the California Department of Fish and Wildlife and the Marine Wildlife Veterinary Care and Research Center for assisting with record archives, alerting us to cases, and sample collection and processing. Jack Ames and Brian Hatfield shared their invaluable insight into the history of sea otter research in California. Thanks also to Dr. Robin McClenahan for giving us a tutorial on sea otter pelage. Special thanks to all those who took the time to note the presence of algae on a sea otter. GWS thanks T. Moore for generating the molecular data for this study and the Natural Sciences and Engineering Research Council Canada, Canada Foundation for Innovation, and New Brunswick Innovation Foundation for funding.

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Received: 19 May 2015 Accepted: 27 August 2015

Supporting Information The following supporting information is available for this article online at http:// onlinelibrary.wiley.com/doi/10.1111/mms.12275/suppinfo. Table S1. BOLD and GenBank accession numbers for red algal samples from five different sea otters submitted to DNA bar code analyses using the markers COI-5P and rbcL.